We have found that phorbol diesters with tumor-promoting activity in mouse skin reversibly inhibit terminal differentiation of erythroleukemia cells and preadipose cells in culture. We propose now to investigate the mechanism of the inhibition and the role that transient inhibition of differentiation by tumor promoters may play in carcinogenesis. A series of differentiating cell lines will be examined to determine if tumor promoters inhibit differentiation only in cultures in which the differentiating cells have a limited lifespan and proliferative capacity (erythroleukemia, preadipocytes, myoblasts) or also inhibit differentiation in cultures in which the differentiating cells retain the potential for continuous proliferation (myeloma, melanoma, colon adenocarcinoma). If the primary action of the promoters is inhibition of expression of "luxury" genes (e.g., hemoglobin), we will determine if the block is at the level of transcription or translation. If the primary effect is to block entrance of cells into G0 (extended G1), the role of polyamines in the block will be examined. We will determine if the frequency of induced mutations in differentiating populations is increased when cells are treated first with a mutagen and then with a promoter. To determine if initiation by a carcinogen can itself inhibit terminal differentiation or can "lock in" the transient inhibition of differentiation by tumor promoters, "normal" differentiating cells (3T3 preadipocytes) will be transformed according to one-stage (carcinogen alone) and two-stage (carcinogen plus promoter) protocols, and cells isolated from transformed foci will be analyzed for the capacity to differentiate. It is becoming increasingly evident that cancer in humans is often the result of the interaction of environmental carcinogens and cocarcinogens such as tumor promoters. These studies on the relationship between cell differentiation and carcinogenesis will help to elucidate the mechanisms by which these interactions lead to neoplasia.